ATP-sensitive potassium (KATP) channels couple cell metabolism to membrane excitability. They play a vital role in energy homeostasis by eliciting physiological response appropriate to metabolic signals. A member of the inwardly rectifying potassium (Kir) channel family, KATP channels are unique in requiring co-assembly of Kir6.2 with a sulfonylurea receptor (SUR), an ABC transporter, for functional expression. In pancreatic - cells, KATP channels formed by Kir6.2 and SUR1 mediate glucose-stimulated insulin secretion. Mutations in Kir6.2 or SUR1 that reduce channel function cause congenital hyperinsulinism whereas those that increase channel function cause neonatal diabetes. Both Kir6.2 and SUR1 play an integral role in the channel's complex gating regulation. The long-term goal of this project is to understand the structure-function relationship of channel proteins with respect to gating. Work in the previous cycle has identified and elucidated the role of several structural features of the pore-forming subunit Kir6.2 in channel gating. In this renewal application, we will address the mechanisms of functional coupling between SUR1 and Kir6.2. SUR1 increases the open probability (Po) of Kir6.2, hypersensitizes Kir6.2 to the inhibitory effect of ATP and stimulatory effect of membrane phosphoinositides, and confers the effects of MgADP as well as the pharmacological agent sulfonylureas and diazoxide on Kir6.2. Functional coupling between SUR1 and Kir6.2 is thus essential to channel gating; yet significant knowledge gaps remain regarding the mechanisms by which SUR1 exerts its multiple effects on Kir6.2. The goal of this renewal application is to elucidate the mechanisms and structural basis that are responsible for functional coupling between SUR1 and Kir6.2. Based on our preliminary data, we propose a unifying hypothesis to explain how SUR1 imposes its multiple effects on Kir6.2 gating. Specifically, we hypothesize that SUR1 confers the intrinsic Po of KATP channels by stabilizing Kir6.2 in the PIP2-bound open state via molecular interactions between residues in the short cytoplasmic loops of TMD0 in SUR1 and those in the N-terminal domain of Kir6.2; and ATP, MgADP and pharmacological agents modulate channel activity in turn by changing the SUR1-Kir6.2 interface to strengthen or weaken channel-PIP2 interactions. We will interweave forward genetics approach employing disease mutations, guided mutagenesis screening, chemical cross-linking and structural modeling approaches to test the hypothesis. The research is innovative because it presents a novel concept to the field. The research is significant from both the human health and basic science standpoints. It will identify new disease mechanisms to directly facilitate diagnosis and treatment of several rare but devastating infant/childhood diseases and will lead to a better understanding of the structure-function relationship of the channel to foster new ideas on how to modulate channel activity to treat diseases caused by channel dysfunction, including type II diabetes. It will also lead to a better understanding of how a silent ABC transporter regulates an ion channel to advance both the ABC transporter and the ion channel fields.